1. Field of the Invention
This invention relates to a method for recovery of antihemophilic factor from human plasma. More particularly, the invention relates to the precipitation of human antihemophilic factor from plasma by a treatment with citric acid, sodium citrate or potassium citrate.
2. Description of the Prior Art
Hemostatis, the biochemistry of blood coagulation, is an extremely complex and as yet not completely understood phenomena whereby normal whole blood and body tissue prevent an excess loss of blood from a ruptured blood vessel. The total mechanism of blood coagulation is affected through the coordinated interaction of biochemical substances contained in three basic physiologic systems; namely, extravascular tissue such as subcutaneous tissue, muscle tissue, and skin; the blood vessel wall; and intravascular components, including blood plasma proteins, blood plasma factors, and platelets.
Hemophilia type A is a genetically induced disease characterized by the loss of clottability of otherwise normal whole blood. It is believed that a cause of hemophilia is the inability of the afflicted individual to synthesize, or produce sufficient quantities of the active form of antihemophilic factor, known as AHG, AHF, Factor VIII or Factor VIII:C (hereinafter AHF) to maintain adequate clotting. Although, at present, hemophilia cannot be cured, it can be treated therapeutically by the administration of AHF to an AHF-deficient patient. The administered AHF is derived from blood obtained from normal and healthy donors and is administered either by transfusion of whole blood or blood plasma, or by infusion of AHF plasma protein concentrate which has been extracted from the plasma of normal human whole blood.
The prior art describes various processes for obtaining AHF from human and animal plasma including separation from other proteins, concentration and purification, such as described in: U.S. Pat. Nos. 4,210,580; 4,361,509; 3,973,002; 4,383,989; and publications: Brinkhous et al, "A New High-Potency Glycine - Precipitated Antihemophilic Factor Concentrate, JAMA, Aug. 26, 1968, Vol 205, No. 9; Newman et al, "Methods for the Production of Clinically Effective Intermediate- and High-Potency Factor VIII Concentrates, British Journ. of Haemat., 1971, (21) 1-20; D. E. G. Austen, "The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose", British Journ. of Haemat., 1979, (43) 669-674; Morgenthaler, "Chromatography of Antihemophilic Factor on Diaminoalkane- and Aminoalkane-Derivatized Sepharose", Thromb. Haemostas. 47 (2) 124-127 (1982); and Faure et al, "Improved Buffer for the Chromatographic Separation of Factor VIII Coagulant; J. Chromatography 257 (1983) 387-391. The object of the prior art is to develop highly purified, highly concentrated AHF preparations from a large quantity of pooled plasma. The high potency AHF concentrates are produced by first separating an AHF rich fraction from pooled plasma by means of Cohn's ethanol fractionation method or from cryoprecipitate obtained by freezing a plasma and then thawing it at low temperature, or from freshly obtained bovine, pig or sheep blood plasma by precipitating the AHF rich fraction with ethanol, ethyl ether, ammonium sulfate, amino acids, glycine and with phosphates or citrates, such as described in U.S. Pat. No. 2,867,567 to Bidwell.
Blood plasma is a scarce and expensive commodity due to limited avaiability of donors, collection and handling difficulties and cost of screening to avoid infectious diseases, such as hepatitis and AIDS. In order to best utilize such a resource and make AHF affordable to hemophiliacs, it is important to prevent losses thereof, during the process of obtaining the purified, therapeutically effective product. One of the most important steps in preventing loss of AHF occurs at separation/precipitation of the AHF-rich proteins from fresh blood or frozen plasma. Depending on the blood type of plasma and the particular methods of separation/precipitation used, recovery of AHF from normal human plasma has generally been in the range of from about 320 to 470 units per liter of plasma with AHF activity ranging from about 650 to about 1,400 units per liter of plasma.
The present invention is directed to a method by which higher recovery of AHF from human plasma is obtained using citric acid, sodium and potassium citrates as precipitants for the AHF-rich fraction.